Pharmaceutical compositions

ABSTRACT

Pharmaceutical compositions containing an iron complex of 3-hydroxy-4-pyrone or of a 3-hydroxy-4-pyrone in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by an aliphatic hydrocarbon group of 1 to 6 carbon atoms, are of value for the treatment of iron deficiency anaemia.

This a rule 62 continuation of application Ser. No. 08/521,281, filedAug. 30, 1995, now abandoned, which is a Rule 62 continuation ofapplication Ser. No. 08/064,800, filed May 21, 1993, now abandoned,which is a Rule 62 continuation of application Ser. No. 07/708,043,filed May 30, 1991, now abandoned, which is a Reissue application ofU.S. Pat. No. 4,834,983, issued May 30, 1989, This application which isa continuation of application of Ser. No. 601,485, filed Apr. 18, 1984,now abandoned, which is a continuation-in-part of application Ser. No.542,972, filed Oct. 18, 1983, now U.S. Pat. No. 4,575,502.

This invention relates to iron compounds for use in pharmaceuticalcompositions for the treatment of iron deficiency anaemia.

An adequate supply of iron to the body is an essential requirement fortissue growth in both man and animals. Although there is normally anample amount of iron in the diet, the level of absorption of iron fromfood is generally low so that the supply of iron to the body can easilybecome critical under a variety of conditions. Iron deficiency anaemiais commonly encountered in pregnancy and may also present a problem inthe newly born, particularly in certain animal species such as the pig.Moreover, in certain pathological conditions there is a mal distributionof body iron leading to a state or chronic anaemia. This is seen inchronic diseases such as rheumatoid arthritis, certain haemolyticdiseases and cancer.

Although a wide range of iron compounds is already marketed for thetreatment of iron deficiency anaemia, the level of iron uptake by thebody of these compounds is often quite low thereby necessitating theadministration of relatively high dosage levels of the compound. Theadministration of high dose, poorly absorbed, iron complexes may causesiderosis of the gut wall and a variety of side effects such as nausea,vomiting, constipation and heavy malodorous stools.

The present invention relates to a group of iron complexes which we haveidentified as being of particular value for use at relatively low dosagelevels in the treatment of iron deficiency anaemia. The hithertounrecognised value of these complexes in such a context, as shown by invivo experiments, is unexpected in view of the well known need forimproved iron compounds for the treatment of iron deficiency anaemia.This is particularly so as among the compounds whose iron complexes areof the most interest for use in pharmaceutical compositions according tothe present invention is a significant number of compounds which arenaturally occurring materials, or are readily derivable from suchmaterials, and which have been known for some time to be capable offorming iron complexes. Furthermore, several of these compounds havepreviously been used in foodstuffs thereby indicating their non-toxicnature and the consequent suitability of pharmaceutical use of theiriron complexes.

According to the present invention a pharmaceutical compositioncomprises an iron complex of 3-hydroxy-4-pyrone or of a3-hydroxy-4-pyrone in which one or more of hydrogen atoms attached toring carbon atoms are replaced by an aliphatic hydrocarbon group of 1 to6 carbon atoms, together with a physiologically acceptable diluent orcarrier.

The iron complex present in the pharmaceutical compositions according tothe present invention preferably contain iron in the ferric state.Although the use of complexes containing iron in the ferrous state maybe considered, such complexes tend to be less stable and are thus ofless interest. The iron complexes are preferably neutral and this isconveniently achieved by complexing with the iron cation the appropriatenumber of anions derived from the hydroxypyrone (through the conversionOH→O—) necessary to produce neutrality. Preferred iron complexes of usein the present invention are thus of the 3:1 form, containing threehydroxypyrone anions complexed with a ferric cation. It will beappreciated, however, that the invention does not exclude the use ofcomplexes of the 1:1 or particularly the 2:1 form, although the 3:1neutral ferric complexes are of especial interest.

The substituted 3-hydroxy-4-pyrones may carry more than one type ofaliphatic hydrocarbon group but this is not usual and, indeed,substitution by one rather than two or three aliphatic hydrocarbongroups is preferred. The aliphatic hydrocarbon groups may be cyclic oracyclic, having a branched chain or especially a straight chain in thelatter case, and may be unsaturated or especially saturated. Groups offrom 1 to 4 carbon atoms are particularly of 1 to 3 carbon atoms are ofmost interest. Alkyl groups are preferred, for example cyclic groupssuch as cyclopropyl and especially cyclohexyl but, more particularlypreferred are acyclic alkyl groups such as n-propyl and isopropyl, andespecially ethyl and methyl. Substitution at the 2- or 6-position is ofespecial interest although, when the ring is substituted by the largeraliphatic hydrocarbon groups, there may be an advantage in avoidingsubstitution on a carbon atom alpha to the

system. The system is involved in the complexing with iron and the closeproximity of one of the larger aliphatic hydrocarbon groups may lead tosteric effects which inhibit complex formation.

Examples of specific compounds whose iron complexes may be used incompositions according to the present invention are shown by thefollowing formulae (I), (II) and (III);

in which R is an alkyl group, for example methyl, ethyl, n-propyl orisopropyl. Among these compounds 3-hydroxy-2-methyl-4-pyrone (maltol;II, R═CH₃) is of most interest, whilst 3-hydroxy-4-pyrone (pyromeconicacid; I), 3-hydroxy-6-methyl-4-pyrone (isomaltol; III, R═CH₃) andparticularly 2-ethyl-3-hydroxy-4-pyrone (ethylpyromeconic acid; II,R═CH₂H₅) are also of especial interest.

In the case of certain of the hydroxypyrones referred to above, forexample maltol, ethylpyromeconic acid and isomaltol, the formation of aniron complex of the compound has been referred to in the literature,although it should be noted that the procedures described in theliterature for the production of such complexes often would not providecomplexes of a form which is preferred for use in the pharmaceuticallycompositions according to the present invention. In the case of otherhydroxypyrones, the iron complexes are novel and are included, per se,by the present invention.

The iron complexes are conveniently prepared by the reaction of thehydroxypyrone and iron ions, the latter conveniently being derived froman iron salt, particularly a ferric halide and especially ferricchloride. The reaction is conveniently effected in a suitable mutualsolvent and water may often be used for this purpose. If desired,however, an aqueous/organic solvent mixture may be used or an organicsolvent, for example ethanol, methanol or chloroform and mixtures ofthese solvents together and/or with water where appropriate. Inparticular, methanol or especially ethanol may be used where it isdesired to effect the separation of at least a major part of aby-product such as sodium chloride by precipitation whilst the ironcomplex is retained in solution.

It should be appreciated that the nature of the iron complex obtained bythe reaction of a hydroxypyrone and iron ions will depend both on theproportion of these two reactants and upon the pH of the reactionmedium. Thus, for the preparation of the 3:1 ferric complex, forexample, the hydroxypyrone and the ferric salt are conveniently mixed insolution in a 3:1 molar proportion and the pH adjusted to a value in therange of 6 to 9, for example 7 or 8. If a similar excess ofhydroxypyrone:iron is employed but no adjustment is made of the acidicpH which results on the admixture of the hydroxypyrone and an iron saltsuch as ferric chloride, then a mixture of the 2:1 and 1:1 complex willinstead by obtained.

Reaction to form the iron complex is generally rapid and will usuallyhave proceeded substantially to completion after 5 minutes at about 20°C., although a longer reaction time may be used if necessary. Followingseparation of any precipitated by-product, such as sodium chloride inthe case of certain solvent systems, the reaction mixture mayconveniently be evaporated on a rotary evaporator or freeze dried toyield the solid iron complex. This may, if desired, be crystallised froma suitable solvent, for example water, an alcohol such as ethanol, or asolvent mixture, including mixtures containing and ether. The presentinvention further includes a process for the preparation of an ironcomplex of 3-hydroxy-4-pyrone or of a 3-hydroxy-4-pyrone in which ahydrogen atom attached to one or more ring carbon atoms is replaced byan aliphatic hydrocarbon group of 1 to 6 carbon atoms, which comprisesreacting said hydroxypyrone with iron ions and isolating the resultantcomplex; such a process being restricted, however, to exclude processesfor the preparation of those particular forms of iron complex ofparticular hydroxypyrones which have already been described in theliterature.

Whilst for some uses it may be appropriate to prepare the iron complexin substantially pure form, i.e. substantially free from by-products ofmanufacture, in other cases, for example with a solid oral formulationas described hereinafter, the presence of by-products such as sodiumchloride may be quite acceptable. In general, however, the neutral 3:1hydroxypyrone:iron(III) complex is of particular interest in a formwhich is substantially free at least from those by-products which arecomplexes containing different proportions of hydroxypyrone and iron, inparticular the 2:1 and 1:1 complexes. Accordingly the present inventionincludes an iron complex, for example the 3:1 hydroxypyrone:iron(III)complex, of 3-hydroxy-4-pyrone or of a 3-hydroxy-4-pyrone in which oneor more of the hydrogen atoms attached to ring carbon atoms are replacedby an aliphatic hydrocarbon group of 1 to 6 carbon atoms, but excluding3-hydroxy-2-methyl-4-pyrone, when in a form substantially free from ironcomplexes of the pyrone containing other proportions of iron. Asindicated hereinafter, it may be advantageous under some circumstancesfor the iron complex to be used in admixture with the free pyrone. It ispossible to produce such a mixture by mixing the two components eitherin the solid form or in solution, followed by isolation of a solidmixture in the latter case when a solid composition is required.However, it may be more convenient to obtain such a mixture by reactinga molar proportion of the pyrone and iron ions of greater than 3:1. Itshould be stressed, however, that the conditions as well as theproportion of reactants used in the reaction are of importance if amixture of the free pyrone and the preferred neutral 3:1 complex is tobe obtained. In particular, as indicated previously, the pH of thereaction mixture is particularly important and, because of this fact,certain prior art procedures concerned with the use of iron pyronecomplexes in food coloring, for example as described in U.S. Pat. No.4,018,907, substantially fail to yield the 3:1 complex even though anexcess of the pyrone is present, owing to the lack of pH control.

Certain hydroxypyrones, such as maltol, are available commercially. Withothers, a convenient starting material in many instances consists ofpyromeconic acid which is readily obtainable by the decarboxylation ofmeconic acid. Thus, for example, pyromeconic acid may be reacted with analdehyde to insert a 1-hydroxy-alkyl group at the 2-position, whichgroup may then be reduced to produce a 2-alkyl-3-hydroxy-4-pyrone. Thepreparation of 2-ethyl-3-hydroxy-4-pyrone, etc., by this route isdescribed in U.S. application Ser. No. 310,141 (series of 1960).

It will be appreciated that these are not the only routes available tothese compounds and their iron complexes and that various alternativesmay be used as will be apparent to those skilled in the art.

The iron complexes may be formulated for use as pharmaceuticals forveterinary or human use by a variety of methods. For instance, they maybe applied as an aqueous, oily or emulsified composition incorporating aliquid diluent, which composition most usually will be employed forparenteral administration, for example intramuscularly, and thereforemay conveniently be sterile and pyrogen free. Oral administration is,however, generally to be preferred for the treatment of iron deficiencyanaemia in humans and the complexes of the present invention may begiven by such a route. Although compositions incorporating a liquiddiluent may be used for oral administration, it is preferred to usecompositions incorporating a solid carrier, for example a conventionalsolid carrier material such as starch, lactose, dextrin or magnesiumstearate. The iron complex will of course be present in such a preferredcomposition in solid form, which form is accordingly a preferred one forthe complex, and such a solid composition may conveniently be presentedas some type of formed composition, for example as tablets, capsules(including spansules), etc.

Although solid compositions are preferred in many applications, liquidcompositions are of interest in certain particular instances, forexample human and veterinary intramuscular administration and veterinaryoral administration as discussed hereinafter. It is often desirable toproduce liquid compositions containing a higher concentration than isreadily obtainable with a purely aqueous composition or indeed onecontaining organic solvents such as simple monohydric alcohols. It hasbeen found that this may be done by the use of solvents containing twoor more hydroxy groups or a hydroxy and an ether group, especially ofglycols or glycol ethers, either in admixture with water or, for bettersolubilisation, alone. The glycol ethers of particular interest are themono-ethers containing as an etherifying group an aliphatic hydrocarbongroup of 1 to 6 carbon atoms as described above, for example a methylgroup, such a glycol mono-ether being methyl ethylene glycol. Ingeneral, however, the glycols themselves are preferred. Examples of suchglycols are the simple dihydroxy alkanes such as ethylene glycol as wellas those more complex compounds comprising two hydroxy groups attachedto a chain containing both carbon and oxygen atoms, such as triethyleneglycol, tetraethylene glycol and polyethylene glycol, for example of4,000 daltons molecular weight. Triethylene glycol and especiallytetraethylene glycol are of particular interest in view of their verylow toxicity. By using such glycols and glycol ethers it is possible toincrease solubility for many complexes to 10 to 20 mg/ml.

In the case of animals, compositions for parenteral administration areof greater interest than with humans. The problems of iron deficiencyanaemia in newly born pigs arise primarily during the first three weeksor so of their life when a very rapid weight gain takes place. Theunusual routes for administration of the iron complexes of the presentinvention to young piglets are parenteral, for example intramuscular, ororal, for example as a liquid preparation “injected” into the mouth.However, an alternative approach is to enhance the iron content of themilk on which the piglets are feeding by treating the mother pig usingoral or parenteral administration, for example with an injectable slowrelease preparation (such an approach may also be of interest in a humancontext). When it is applicable to feed piglets on foodstuffs other thanthe milk of the mother pig, it may also be possible to effect thepharmaceutical administration of the iron complex in this otherfoodstuff.

Other forms of administration than by injection or through the oralroute may also be considered in both human and veterinary contexts, forexample the use of suppositories or pessaries for human administration.

Compositions may be formulated in unit dosage form, i.e. in the form ofdiscrete portions containing a unit dose, or a multiple or sub-unitdose. Whilst the dosage of hydroxypyrone iron complex given will dependon various factors, including the particular compound which is employedin the compositions, it may be stated by way of guidance thatmaintenance at a satisfactory level of the amount of iron present in thehuman body will often be achieved using a daily dosage, in terms of theiron content of the compound, which lies in a range from about 0.1 to100 mg and often in a range from 0.5 to 10 mg, for example 1 or 2 mg,veterinary doses being on a similar g/Kg body weight ratio. However, itwill be appreciated that it may be appropriate under certaincircumstances to give daily dosages either below or above these levels.In general, the aim should be to provide the amount of iron required bythe patient without administering any undue excess and the properties ofthe pharmaceutical compositions according to the present invention areparticularly suited to the achievement of this aim. Similarly, theconcentration of iron in the pharmaceutical composition in the form ofthe hydroxypyrone complex may vary quite widely, for example over arange from about 0.001 to about 20% w/w. However, it is more usual forthe concentration to exceed 0.01% w/w and it may often exceed 0.05 or0.1% w/w, whilst a more usual limit for the upper end of the range isabout 13% w/w. A common range of concentration is 0.05 to 5% w/w, forexample 0.2 to 0.5, 1 or 2% w/w.

Where desired, more than one hydroxypyrone iron complex as describedabove may be present in the pharmaceutical composition or indeed otheractive compounds having the ability to facilitate the treatment ofanaemia, such as folid acid. Another additional component which may beincluded in the composition, if desired, is a source of zinc. Ironcompounds used in the treatment of iron deficiency anaemia can inhibitthe mechanism of zinc uptake in the body and this can cause serious sideeffects in the foetus when treating anaemia is in a pregnant female. Itis believed, however, that the iron complexes of the present inventionhave a further advantage in that they either do not have this effect orexhibit the effect at a lower level than the compounds at present usedin the treatment of anaemia. Accordingly, it may often be the case thatthe level of zinc providing compound added to the composition may notrequire to be high or, with preferred formulations of the ironcomplexes, may be dispensed with altogether.

Although certain of the iron complexes, for example iron maltol, havepreviously been proposed for use as colouring agents in foodstuffs, ithad never previously been appreciated that they have any therapeutic useand the conditions proposed for the use of such complexes as colouringagents would not generally be such as to lead to any significantphysiological effect. Accordingly the present invention includes an ironcomplex of 3-hydroxy-4-pyrone or of a 3-hydroxy-4-pyrone in which one ormore of the hydrogen atoms attached to ring carbon atoms are replaced byan aliphatic hydrocarbon group of 1 to 6 carbon atoms, for use inmedicine, particularly in the treatment of iron deficiency anaemia.

We have found that the iron complexes described herein are particularlysuited to the treatment of iron deficiency anaemia, both in humans andalso in a veterinary context and particularly for the treatment ofvarious mammalian species, especially pigs. Thus, the chelating agentswhich they contain, and particularly maltol, have a high affinity foriron (log β₃=30 for maltol) but a lower affinity for copper (II), zinc(II), calcium and magnesium. Both the high affinity of maltol for ironand its low affinity for calcium are reflected in its K_(sol) value {logK_(sol) is defined as being equal to log α_(Fe(L)n)+21−[pK_(sp)+n loga_(L)(H⁺)+m log a_(L)(Ca⁺⁺)] where log β_(Fe(L)n) is the cumulativeaffinity constant of the ligand in question for iron (III), pK_(sp) isthe negative logarithm of the solubility product for Fe(OH)₃ and has avalue of 39, n and m are the number of hydrogen and calcium ions,respectively, which are bound to the ligand, and a_(L)(H⁺) anda_(L)(Ca⁺⁺) are the affinities of the ligand for hydrogen ions andcalcium ions, respectively}. In order to solubilise iron(III) hydroxide,log K_(sol) must be greater than 0. The value of K_(sol) for maltol is0.8 and this is also sufficiently large to prevent appreciablecompetition from phytate, phosphate, thiols and other potential ligandslikely to occur in the intestinal lumen. In order to exchange ironefficiently with transferrin, the log K_(sol) value should be close tothat of apotransferrin, which is 6.0, so that maltol is also suitable inthis respect, Moreover, although the neutral 3:1 maltol:iron(III)complex is thermodynamically stable (thermodynamic stabilityconstant=30) it is also extremely labile and is therefore able to donateiron to high affinity sites, such as those found in apotransferrin. Thehalf life for the exchange of iron(III) between the maltol complex andapotransferrin is 1 minute whereas, by contrast, the correspondingfigure for the complex of EDTA with iron(III) is 4 days.

It will be appreciated, however, that in addition to possessingproperties such as those described above for iron maltol, a compoundwhich is to act as a source of iron though oral administration isrequired to to show a high level of membrane permeability. An indicationof the properties of a compound in this respect is provided by the valueof the partition coefficient (K_(part)) obtained on partition betweenn-octanol and Tris hydrochloride (20 mM, pH 7.4; Tris representing2-amino-2-hydroxymethylpropane 1,3-diol) at 20° C. and expressed as theratio (concentration of compound in organic phase)/(concentration ofcompound in aqueous phase). The value of K_(part) for the neutral 3:1maltol:iron(III) complex is 0.5, which is well placed in the preferredrange of 0.2 to 1.0 and compares favourably with the figures of 0.001and 0.0015 for the EDTA:iron(III) complex and iron(III) ascorbate,respectively.

The value of the iron complexes of the present invention is configuredby various in vitro and in vivo tests. Thus, their ability to permeatebiological membranes is confirmed in practice by tests of the ability ofthe ⁵⁹Fe labelled iron complexes to permeate erythrocytes. Moreover,iron complexes of the present invention have been found to exhibit ahigh level of efficiency in promoting iron uptake, as measured in therat small intestine, as compared with a range of other iron complexescurrently marketed for the treatment of iron deficiency anaemia. In vivoexperiments in the cat and rat have confirmed the value of iron maltolcompounds as a source of iron, the iron uptake obtained either onintravenous administration or on direct administration into the smallintestine being markedly superior to that obtained with commerciallyavailable iron compounds such as iron sulphate, iron EDTA and irongluconate. It was found from these experiments that the iron was notexcreted to any significant extent in the urine but became generallydistributed throughout the body, the complexes donating iron totransferrin to an equilibrium level one they are present in thebloodstream.

Certain aspects of their formulation may enhance the activity of thecomplexes in particular contexts. Thus, although the neutral 3:1 ferriccomplexes are stable over a wide pH range from about 4 or 5 up to 10,they will dissociate at the pH values of less than 4 prevailing in thestomach to form a mixture of the 2:1 and 1:1 complex together with thefree hydroxypyrone, and it has been found that the blood levels of ⁵⁹Feachieved on administration of the 3:1 complex into the small intestineare much higher than when administration is made into the stomach.However, when the stomach contents is flushed to the small intestine inin vivo cat experiments an increase of iron uptake occurs almostimmediately. The undesirable effects of this dissociation on iron uptakemay be countered by using one or more of the following procedures in theformulation of the iron complex. Firstly, one of several variations maybe employed which avoid or reduce exposure of the iron complex to theacidic conditions of the stomach. Such approaches may involve varioustypes of controlled release system, ranging from one, which may forexample be based on a polymer, which simply provides a delayed releaseof the complex with time, through a system which is resistant todissociation under acidic conditions, for example by the use ofbuffering, to a system which and is biased towards release underconditions such as prevail in the small intestine, for example a pHsensitive system which is stabilised towards a pH of 1 to 3 such asprevails in the stomach but not one of 7 to 9 such as prevails in thesmall intestine. Since the pH of the stomach is higher after a meal, itmay be advantageous, whatever method of formulation is used, toadminister the iron complexes at such a time.

A particularly convenient approach to a controlled release compositioninvolves encapsulating the iron complex by a material which is resistantto dissociation in the stomach but which is adapted towards dissociationin the small intestine (or possibly, if the dissociation is slow, in thelarge intestine). Such encapsulation may be achieved with liposomes,phospholipids generally being resistant to dissociation under acidicconditions. The liposomally entrapped 3:1 iron(III) complexes cantherefore survive the acid environment of the stomach withoutdissociating to the 2:1 and 1:1 complexes, and the free hydroxypyrone.On entry into the small intestine, the pancreatic enzymes rapidlydestroy the phospholipid-dependent structure of the liposomes therebyreleasing the 3:1 complex. Liposome disruption is further facilitated bythe presence of bile salts. However, it is usually more convenient toeffect the encapsulation, including microencapsulation, by the use of asolid composition of a pH sensitive nature.

The preparation of solid composition adapted to resist dissociationunder acidic conditions but adapted towards dissociation undernon-acidic conditions is well known in the art and most often involvesthe use of enteric coating, whereby tablets, capsules, etc, or theindividual particles or granules contained therein, are coated with asuitable material. Such procedures are described, for example, in thearticle entitled “Production of enteric coated capsules” by Jones inManufacturing Chemist and Aerosol News, May 1970, and in such standardreference books as “Pharmaceutical Dosage Forms”, Volume III byLiebermann and Lackmann (published by Marcel Decker). One particularmethod of encapsulation involves the use of gelatine capsules coatedwith a cellulose acetate phthalate/diethylphthalate layer. This coatingprotects the gelatin capsule from the action of water under the acidconditions of the stomach where the coating is protonated and thereforestable. The coating is however destablished under the neutral/alkalineconditions of the intestine where it is not protonated, thereby allowingwater to act on the gelatin. Once released in the intestine the rate ofpermeation of the intestine wall by the water soluble 3:1 iron(III)complex is relatively constant irrespective of the position within theintestine, i.e. whether in the jejunum, ileum or large intestine. Otherexamples of methods of formulation which may be used include the use ofpolymeric hydrogel formulations which do not actually encapsulate theiron complex but which are resistant to dissociation under acidicconditions.

A second approach to countering the effect of the acidic conditionsprevailing in the stomach involves formulation of the complex in thepharmaceutical composition together with the metalfree hydroxypyronefrom which it is derived. The dissociation of the neutral 3:1 ferriccomplex, for example, involves various equilibria between this complex,the 2:1 and 1:1 complexes, and the metal-free compound, so that thepresence of the latter will inhibit this dissociation. Any proportion ofthe free compound can be advantageous in this context but little furtheradvantage accrues from increasing the proportion beyond a certain level.A preferred range for the molar proportion of the free compound presentin compositions according to the present invention is thus from 0 to 100moles of free hydroxypyrone:1 mole of iron complex, particularly of theneutral 3:1 iron(III) complex. Conveniently, a proportion of up to nomore than 20, 30 or 50 moles:1 mole is used with a lower level of 0.5, 1or 2 moles:1 mole. Although to obtain a marked effect upon dissociationof the iron complex a proportion of at least 5 or 10 moles:1 mole isusually employed, it should be emphasised that even a molar ratio suchas 1:1 will achieve a noticeable degree of acid stabilisation of theiron complex. Thus although a range of, for example, from 10 moles:1mole to 20 moles:1 mole of metal-free hydroxy pyrone:iron complex willoften be suitable to produce a marked effect, a range of, for example, 3or even 1 mole:1 mole to 10 moles:1 mole will still produce a worthwhileeffect without requiring administration of the larger amounts of thehydroxy pyrone. It will be appreciated that a mixture of thehydroxypyrone and iron complex in at least certain of the proportionsdescribed above is novel for the various hydroxypyrones, irrespective ofthe particular form of the iron complex, and that such novel mixturesare included by the present invention. The use of such a mixture is animportant feature of the present invention since in principle it canenable one to obtain almost quantitative uptake of iron from thecomplex.

The use of an uncompleted hydroxypyrone in admixture with its ironcomplex may also have another advantage in addition to the prevention ofdissociation of the iron complex under acidic conditions. Thus, incertain pathological conditions there may be an excess of iron depositedat certain sites even though the patient exhibits an overall anaemia. Inthese patients the use of such a mixture has the advantage that the ironcomplex will remedy the overall anaemia whilst the free hydroxypyronewill act to remove iron from pathological to physiological sites.However, although it is preferable for the hydroxypyrone present in aniron donor to be rapidly metabolized in order that iron may beefficiently transferred to the binding proteins and eventually to theiron requiring mechanisms within the body, it is preferable for ahydroxypyrone being used as an iron remover not to be rapidlymetabolized so that it remains in the system, taking up iron, for anextended period. Thus, for example, maltol is rapidly metabolized and istherefore particularly suited for use as an iron complex, but for thissame reason it is not appropriate for use in the free form. It is alsothe case that different compounds may function more efficiently eitherin the free form as an iron remover of in complex form as an iron donorfor quite other reasons. Accordingly the present invention includes amixture of an iron complex of a 3-hydroxy-4-pyrone or of a3-hydroxy-4-pyrone in which one of the hydrogen atoms attached to ringcarbon atoms are replaced by an aliphatic hydrocarbon group of 1 to 6carbon atoms, together with a different 3-hydroxy-4-pyrone.Alternatively, the different 3-hydroxy-4-pyrone may be replaced by aquite different form of iron chelating agent. Examples of such orderiron chelating agents which may be used include the substituted3-hydroxypyrid-2-ones and -4-ones, and 1-hydroxypyrid-2-one andsubstituted 1-hydroxypyrid-2-ones (and salts of these various pyridoneswith a physicologically acceptable cation) described in copending U.S.patent application Ser. Nos. 592,271 (now U.S. Pat. No. 4,585,780)478,493 and 651,772 (now U.S. Pat. No. 4,587,240).

When a free hydroxy-4-pyrone, hydroxypyrid-2-one, hydroxypyrid-4-one, orother iron chelating agent is present in admixture with the iron complexof a hydroxy-4-pyrone for the purpose of acting as an iron remover, thenthe amount of this agent used may be different than when a freehydroxypyrone necessarily corresponding to that present in the ironcomplex is present primarily to prevent dissociation. Thus the dailydosage of the iron complex may be as above and the daily dosage of thefree iron chelating agent, particularly when this is a hydroxypyrid-2-or -4-one or a 1-hydroxy-pyrid-2-one, may be that quoted in thecopending applications referred to above, i.e. about 0.1 g to 5 g forhuman use, particularly 0.5 g to 2 g, from which it will be seen thatthe proportion of iron complex and free iron chelating agent used insuch a context may extend across a wide range but referred amounts ofthe free iron chelating agent to be higher than when this is necessarilya hydroxypyrone.

It will be appreciated that the present invention also includes a methodfor the treatment of a human or other mammalian patient which comprisesadministering to said patient an iron complex of 3-hydroxy-4-pyrone orof a 3-hydroxy-4-pyrone in which one or more of the hydrogen atomsattached to ring carbon atoms are replaced by an aliphatic hydrocarbongroup of 1 to 6 carbon atoms in order to effect an increase in thelevels of iron in the patient's bloodstream.

In addition to the pharmaceutical uses of the iron complexes discussedabove they are also of potential interest as a source of iron in variousother contexts including cell and bacterial growth, plant growth, andthe control of iron transport across membranes.

This invention is illustrated by the following Examples:

EXAMPLES Example 1 The Preparation of Iron Maltol

A chloroform solution of maltol is mixed with a 1M solution of ferricchloride in ethanol to provide a 3:1 molar ratio of maltol:iron in themixture. After 5 minutes at 20° C., a 10 molar excess of solid sodiumcarbonate is added and the mixture is stirred for 10 minutes. Themixture is then filtered and the solvent evaporated to give the neutralcomplex containing maltol and the ferric cation in 3:1 proportion.Recrystallisation of the 3:1 complex from the ethanol gives wine redneedle crystals in an essentially quantitative yield, m.p. 275°, ν_(max)(nujol) 1600 cm⁻¹.

The use of an excess of maltol above the 3:1 molar ratio leads to anessentially quantitative yield of a solid mixture of the excess maltoland the 3:1 iron maltol complex on rotary evaporation, this mixture notbeing deliquescent.

The partition coefficient K_(part) (concentration inn-octanol/concentration in aqueous phase) between n-octanol and Trishydrochloride (20 mM, pH 7.4) of maltol and of its 3:1 iron complex ismeasured at 10⁻⁴ M by spectrophotometry. Acid washed glassware is usedthroughout and, following mixing for 1 minute, the aqueous/n-octanolmixture is configured at 1000 g for 30 seconds. The two resulting phasesare separated for a concentration determination by spectrophotometry oneach. For maltol, the range 220-340 nm is used for the concentrationdetermination whilst for the complex the range 340-640 nm is used.Typically, a value of 0.66 is obtained for maltol and of 0.50 for itsiron complex, whilst comparative experiments on iron(III) EDTA andiron(III) ascorbate give much smaller values of 0.001 and 0.0015,respectively.

The ability of the iron complex of maltol to bind to haemoglobin isinvestigated by studying the elution profile of a ⁵⁹Fe label when amixture of haemoglobin and the ⁵⁹Fe-labeled complex (at 1 mMconcentration) in NaCl (130 mM) buffered to pH 7.4 by Tris hydrochlorideis applied to a PH-10 column (Sephadex G-10 gel permeationcolumn—Pharmacia). Typically, no evidence if found for binding of thecomplex to haemoglobin which is an advantageous finding since suchbinding reduces availability of the iron.

The ability of the iron complex of maltol to bind to bovine serum albmen(BSA) is investigated through a similar procedure in which the complexis applied to a column with BSA rather than haemoglobin. The ironcomplex also shows little ability to bind to BSA.

Example 2 In vitro Tests on Permeation of Iron Complexes into HumanErythrocytes

The accumulation of iron by human erythrocytes which are associated withthe iron compiles of maltol described in Example 1, and various otheriron compounds by way of comparison, was studied by incubating theerythrocytes for 1 hour at 37° C. in a medium consisting of the ⁵⁹Felabelled iron compound in aqueous sodium chloride (130 mM) buffered to apH of 7.4 by Tris hydrochloride. Following this period of incubation analiquot of the erythrocyte/medium mixture was placed above a layer ofsilicone oil (ρ=1.06) and the erythrocytes separated by centrifugationthrough the oil. The ⁵⁹Fe levels associated with the erythrocytes andthe incubation medium were then counted and presented as a distributionratio (concentration in erythrocytes/concentration in medium). Theratios obtained for the various iron compounds after incubation for 1hour are shown in Table 1 where it will be seen that the uptake of ironis clearly much greater with the iron maltol complex than with the othercompounds. Values of less than 0.1 are probably associated with bindingto the external surface and do not represent transmembrane movement ofiron. Moreover, although a period of 1 hour was employed in order tofacilitate monitoring of the more slowly permeating iron compounds, theuptake of iron maltol complex reached equilibrium at the level shownafter about 15 minutes.

TABLE 1 Concentration Distribution Compound (mM) ratio Fe^(III)(maltol)₃ 3 1.60 Fe^(II) gluconate 1 0.08 Fe^(III) ascorbate 1 0.12Fe^(III) citrate 1 0.05 Fe^(III) EDTA 1 0.05

When the above described procedure was applied using ratios of maltol toiron of less than 3:1 larger apparent distribution ratios were observedthan 1.60. However, this is explained by the non-specific binding of thepositively charged 2:1 and 1:1 maltol:iron complexes to the surface ofthe erythrocytes which possesses a net negative charge, being rich inboth phosphate and sulfate moieties. Experiments to determine thepercentage of ⁵⁹Fe associated with erythrocyte ghosts after lysisconfirm this hypothesis. In one experiment, lysis was initiated by asmall volume of 10% v/v Triton X100 and in a second experiment by a 10fold excess of water. In each case the resulting ghosts were centrifugedthrough silicon oil (ρ=1.02) and, as will be seen from Table 2, verylittle of the 3:1 maltol:iron complex was found to be bound to themembranes, in contrast with the situation with the 2:1 and 1:1complexes. Such binding is of course undesirable as the complex islikely to remain tightly bound to the membrane by electrostaticinteractions and not be transmitted across it.

TABLE 2 Iron associated with Ratio of with ghosts (%) maltol:iron Tritonlysis Hypotonic lysis 0:1 100 — 1:1 55 63 2:1 22 39 3:1 <1 <5

Example 3 In vitro Tests on Permeation of Rat Jejunal Sac by IronComplexes

The iron uptake into the several space of the rat jejunal sac wascompared for the iron complex of maltol described in Example 1 andvarious other iron compounds by way of comparison. Rats (male SpragueDawley, 60 g) were killed and the jejunum removed, everted and cut intothree segments (4 cm length). The segments were tied at both ends,filled with Krebs Ringer buffer (0.2 ml) and incubated in Krebs Ringerbuffer containing the appropriate ⁵⁹Fe compound at 37° C. for periods upto 90 minutes. The contents of the sac were counted for ⁵⁹Fe andmeasured spectrophotometrically.

The results obtained for the iron maltol complex and for 6 other ironcompound which are each contained in preparations marketed for thetreatment of iron deficiency anaemia are shown in Table 3, the ironuptake for each at 15 and 60 minutes after the initiation of theexperiment being shown relative to that for ferric chloride as 1. Itwill be seen that the iron maltol complex provides a level of ironuptake which is significantly higher than the levels observed for any of6 compounds in current use for the treatment of iron deficiency anaemia.The uptake of the iron maltol complex was linear for a period 90minutes. Moreover, the uptake increased linearly as the concentration ofthe complex was increased over a range of 0.5 to 10 mM, so it does notshow saturation kinetics and the process is thus non-facilitated andtherefore should occur in all natural membranes.

TABLE 3 Relative iron uptake Compound 15 minutes 60 minutes FeCl₃ 1 1Fe^(III) (maltol)₃ 40 5.8 Fe^(II) sulphate 2.4 1.4 Fe^(II) fumarate 4.01.8 Fe^(II) gluconate 1.6 0.8 Fe^(II) succinate 2.0 1.0 Fe^(III)ascorbate 0.4 0.8 Fe^(III) citrate 2.0 1.8

The procedure described above was used to compare the uptake of ironfrom buffer containing differing molar proportions of maltol:iron. Theresults obtained are presented in Table 4 which shows the amount of timetransferred via the maltol complex into the serosal contents of the sac,the basal uptake of iron measured in a control experiment beingsubtracted in each case. It will be seen that the amount of irontransferred in the case of a 3:1 molar proportion of maltol:iron(III) ismuch higher than in the other two cases and, moreover the low, butsignificant level of iron uptake observed in the case of a 2:1 ratio issubstituted to the proportion of the 3:1 complex (containing 13% of thetotal iron) present under these conditions.

TABLE 4 Maltol/iron Iron uptake (molar ratio) (n mole) 1:1 1.6 2:1 4.03:1 30.0

Example 4 In vivo Test of Action of Iron Compounds in the Rat

The action of the iron complex of maltol described in Example 1 wascompared with that of iron(II) sulphate, iron(III) EDTA (1:1 gmolarratio) and iron(II) gluconate.

Groups of rats (300-350 g) were anaesthetised with nembutal (0.25 ml)and then with ether. A mid-line incision was made and the ⁵⁹Fe labelledsample (100 μg Fe 10 μCi) was passed into the lumen of the duodenum viaa small incision. The abdominal well was then closed with a suture. Theanimals were sacrificed 1, 2, 4 and 6 hours after the administration ofthe compound and the various organs were monitored for their ⁵⁹Fecontent. The data is presented as histograms in FIGS. 1 to 4 whichrelate to iron maltol, iron sulphate, iron EDTA and iron gluconate,respectively, and shown the levels of ⁵⁹Fe in cpm after various timeintervals for the different organs, the data in each case representing amean of the results for three individual animals. In the case of thedata for blood and sternum (bone marrow) the counts given are cpm/ml andcpm/g respectively, whilst in all other cases they are the total cpmcounts. The various histograms have been normalised and consequently aredirectly comparable.

A comparison between FIGS. 1 and 2 shows that the neutral 3:1maltol:iron(III) complex is markedly superior to iron(II) sulphate forthe introduction of iron via the rat intestine. The gut washings (whichcontain non-absorbed iron) show a much lower level of counts for themaltol complex, and the counts associated with the gut wall, liver,blood, bone marrow and spleen are correspondingly greater. It is clearfrom FIG. 1 that ⁵⁹Fe associated with maltol enters the intestine wallvery rapidly and from there it is efficiently removed by the bloodsupply. Iron is deposited in the bone marrow continuously throughout the6 hour period at an apparently constant rate.

The maltol complex is also more efficient than iron(III) EDTA as shownby FIG. 3. With the later complex, the gut washings remain high for 4hours and may be pressured to decrease only due to the effect of naturalbowel movements translocating material from the portion underinvestigation to lower portions of the intestine. The levels in theintestine wall and blood are extremely low. Although iron is transferredto both bone marrow and spleen, this is at reduced rates as compared tothose obtained with the maltol complex. As shown by FIG. 4, iron(II)gluconate proved more effective than the sulphate or the EDTA complex,although deposition in the gut wall was less than that observed with themaltol complex. The decrease was reflected in the lower levels of ⁵⁹Fein both bone marrow and the spleen, the difference being particularlymarked after 6 hours. In view of the much higher levels of ⁵⁹Fe trappedin the intestine wall in the case of the maltol complex, it may bepredicted that this compound facilitates a more prolonged supply of ironthan iron(II) gluconate.

This test illustrates the superiority of the neutral 3:1maltol:iron(III) complex as compared with three commonly used “solubleiron” preparations for the movement of iron across the rat jejunal wallinto the blood circulation, the iron maltol being very rapidly removedfrom the lumen of the intestine.

Example 5 In vivo Test of Action of Iron Complex in the Cat

The action of the iron complex of maltol described in Example 1 wascompared with that of iron(III) EDTA (1:1 molar ratio) which is one ofthe iron compounds currently marketed for the treatment of irondeficiency anaemia. Cats were anaesthetised with chloralase (60 mg/kg)and pentobarbitone sodium (60 mg/kg) (i.p.), having been kept free offood for 18 hours. In each animal the trachea was cannulated to maintaina clear airway and to allow positive pressure artificial respiration ifnecessary. The left femoral vien was cannulated for the intravenousadministration of drugs and physiological saline solution. Arterialblood pressure was monitored by a Washington pressure transducer througha cannula inserted into the femoral artery of the right hind leg.Arterial blood samples were taken at appropriate intervals from a shortcannula inserted into an external carotid artery. Body temperature wasmonitored with a rectal thermometer. Each animal was given heparin (1000iu/kg) as anticoagulant and additional small amounts of pentobarbitonesodium if needed to maintain a satisfactory level of anaesthesia.

If those animals where the iron compounds were to be administered intothe duodenum, a mid-line incision was made in the abdomen to reveal theintestines. A cannula was then inserted through a small cut such thatits tip rested approximately 5 cm below the opening of the bile duct.The cannula was then sutured in place and the abdominal wall closed withstitches.

The iron maltol complex (100 μg Fe) alone (3:1 molar ratio ofmaltol:iron) and together with a large excess of maltol (40:1 molarratio of maltol:iron) was injected intravenously in separate experimentsand 0.25 ml samples of blood were taken in intervals. The apparentvolume of distribution of the compound was calculated by extrapolationof the log-linear blood concentration curve to zero. (The volumecorresponds to a value between that of the total extracellular space ofthe animal and the blood volume.) Elimination of ⁵⁹Fe from the bloodfollowed first order kinetics with a rate constant of −0.022/minute inthe presence and absence of excess maltol, as illustrated in FIG. 5which shows the ⁵⁹Fe level in the blood in cpm/0.25 ml plotted againsttime in the case of one typical experiment of each type in an individualcat.

The distribution of ⁵⁹Fe in the tissues of the animal after the sameintravenous experiment to which FIG. 5 relates (the lower end of theordinate in this Figure represents the background level) wasinvestigated and the typical results are shown in Table 5. The amount of⁵⁹Fe administration in this experiment was 4 μCi or 2.2×10⁶ cpm. It willbe seen that approximately 10% of the dose was located in the combinedtissue of the heart, liver and spleen. As less than 0.2% of the dose waslocated in the urine, the bulk (approximately 90% of the ⁵⁹Fe was almostcertainly directed to the bone marrow and extremely high levels werefound to be located in the sternum.

As indicated previously, the maltol complex is able to donate ironrapidly to transferrin and it is hypothesised that such an exchangeoccurs as soon as the complex is delivered to the plasma; and that theinitial plateau (represented in FIG. 5 by a dotted line) representssaturation of the plasma transferrin pool with ⁵⁹Fe. When there is netdonation of ⁵⁹iron from the plasma into the organ is of the animal, theblood levels of radioactivity begin to fall, the major route of transferof iron bound to transferrin being to the bone marrow, liver and spleen.Binding of ⁵⁹Fe to transferrin prevents its excretion in the urine.

TABLE 5 (Iron maltol, i.v.) Sample Net ⁵⁹Fe Net total ⁵⁹Fe Total tissueweight content content Tissue weight (g) (g) (cpm/g) (cpm) Heart 14.40.91 490 7,056 Liver 105 1.3 510 53,550 Spleen 8.4 0.86 14,890 125,076Kidney 12.2 1.05 546 6,661 Skeletal muscle — 1.85 0 0 Sternum — 1.23,200 — (bone marrow) Urine — 1 152 <3,000

Identical experiments carried out with ⁵⁹Fe labelled iron(III) EDTA gavea entirely different picture as will be seen for the results of atypical experiment illustrated in FIG. 6 (in which the lower end of theordinate represents the background level) and Table 6 (the amount of⁵⁹Fe administered in this experiment was 2 μCi but the figures given inthe table have been adjusted to correspond to a dosage of 2.2×10⁶ cpm inorder to facilitate comparison with Table 5). In this experiment theradioactivity in the blood showed no initial plateau. Instead, loss ofradioactivity followed at least a two-component process such that alarge amount found its way to the urine rather than to the issues. Therate constant of the elimination from the blood to the linear phase ofthe regression was 0.023/minute. The concentration of radioactivity inthe kidney and urine, and not in the bone marrow or spleen, wouldindicate that iron in this form does not appear to be able to attach totransferrin in the plasma and protect itself from urinary excretion. Thecombined tissue of heart, liver and spleen contained only 1% of theoriginal dose at the end of the experiment, whereas the urine containedover 50%. This is in accord with the fact that EDTA does not exchangeiron with transferrin rapidly.

TABLE 6 (Iron EDTA, i.v.) Sample Net ⁵⁹Fe Net total ⁵⁹Fe Total tissueweight content content Tissue weight (g) (g) (cpm/g) (cpm) Heart 15.51.01 209  3,248 Liver 75   1.21 261 19,600 Sternum — 0.28 1,164 — (bonemarrow) Spleen 11.1 0.89 162  1,814 Kidney 19.2 1.47 1,134 21,770Skeletal muscle — 2.59 95 — Urine 19 ml 2 ml 62,156 1,180,900  

The iron maltol complex (100 μg Fe) was also administered to theduodenum of the cat in the presence of a 40 fold excess of maltolfollowed by 5 ml of 150 ml tris hydrochloride buffer (pH 7.4). In thiscase the ⁵⁹Fe content of the blood, as shown in FIG. 7, reaches amaximum level 2 hours after the initial administration (the readingsstart at about 300 cpm/0.5 ml which represents the background reading).The distribution of ⁵⁹Fe in the tissues of the animal after the sameduodenal experiment to which FIG. 7 relates were investigated and thetypical results are shown in Table 7. The amount of 59Fe andadministered in this experiment was 10 μCi or 5.327×10⁶ cpm into a 2.9kg cat. It will be seen that the distribution of the ⁵⁹Fe after 4 hourswas similar to that after intravenous infusion, with low levels in thekidney and urine and high levels in both the spleen and bone marrow.

TABLE 7 (Iron maltol, per duodenum) Sample Net ⁵⁹Fe Net total ⁵⁹Fe Totaltissue weight content content Tissue weight (g) (g) (cpm/g) (cpm) Heart14  0.633 50 1,106 Liver 81 1.45 400 32,400 Spleen 12.7 1.19 3,78348,047 Kidney 14.4  0.835 79 1,138 Sternum 10 1.26 790 7,905 (bonemarrow) Bile ˜5 ml 1 ml 2,200 ˜11,000 Urine ˜10 ml 1 ml 22 ˜220

When ⁵⁹Fe labelled iron(III) EDTA was administered duodenally in thesame manner, the plasma levels of radioactivity hardly exceeded thebackground level and are therefore not illustrated in a Figure. Thedistribution of ⁵⁹Fe in the tissues of the animal after the sameduodenal experiment were investigated and the typical results are shownin Table 8. The amount of ⁵⁹Fe administered in this experiment was 10μCi or 2.65×10⁶ cpm into a 2/9 kg cat. It will be seen that, althoughsome ⁵⁹Fe entered the tissues, rather low levels were detected in thespleen and bone marrow (sternum) whereas a large proportion of the dosewas located in the urine.

TABLE 8 (Iron EDTA, per duodenum) Sample Net ⁵⁹Fe Net total ⁵⁹Fe Totaltissue weight content content Tissue weight (g) (g) (cpm/g) (cpm) Heart15.3 1.18 188 2,878 Liver 59.3 0.78 499 29,574 Kidney 11.3 0.90 1,76219,913 Spleen 4.4 0.42 200 880 Sternum — 0.78 917 — (bone marrow)Skeletal muscle — 1.48 117 — Urine 15 ml 5 ml 36,306 544,596

Example 6 Polymer Formulation of Iron Maltol

A solution of ferric chloride (concentration between 1 and 5% w/v),together with maltol in a weight ratio of 8 parts by weight of maltol to1 part by weight of ferric chloride, is prepared in a 4.5:4.5:1 v/v/vmixture of chloroform:methanol:water. Sodium carbonate is added in a 10molar excess over the iron content in order to remove hydrochloric acidand the precipitated NaCl and Na₂CO₃ are filtered off. The preparationis contacted with a cross-linked polyethylene glycol hydrogel materialto effect take up the solution by the polymer and provide a polymerformulation of iron maltol in the presence of excess maltol.

Example 7 Capsule Formulation of Iron Maltol

A preparation of iron(III) maltol in admixture with maltol (containing 1part by weight of iron to 10 parts by weight of maltol) is obtained bythe addition of a 1M ethanolic solution of ferric chloride to amethylene chloride solution of appropriate amount of maltol, followedafter 5 minutes at 20° C. by treatment with a 10 molar excess of solidcarbonate, stirring for 10 minutes, filtration and evaporation of thesolvents.

The resulting solid iron(III) maltol preparation is divided into 50 mgquantities and added to standard gelatine capsules (16×5 mm), eachcapsule containing 5 mg of iron. The capsules are then coated with acellulose acetate phthalate/diethylphthalate layer (6 mg coat per cm² ofcapsule surface) in a small scale procedure analogous to the proceduredescribed by Jones, ibid. A proportion of the capsules are treated toadd a second similar coating.

Such capsules are resistant to dissociation in the stomach but willundergo dissociation in the intestine. Thus, when treated at 37° C. withdilute aqueous hydrochloric acid (pH 2.0) the singly coated capsules aretypically stable for 30 minutes but in Krebs Ringer bicarbonate solution(pH 7.4) at 37° C. they dissociate to release the iron complex within 1minute. The doubly coated capsules a typically stable at pH 2.0 for 20hours, again dissociating within 1 minute at pH 7.4.

Example 8 Liposome Formulation of Iron Maltol

(A) a solution of egg yolk phosphatidyl chlorine⁽¹⁾ (40 mg) andcholesterol (40 mg) in chloroform (1 ml) is rotary evaporated in a 50 mlround bottomed flash to form a thin lipid film. An aqueous solution ofthe 3:1 neutral iron(III) maltol complex (6 ml, 1 mg/ml) is added to theflask and the mixture is vibrated for 15 minutes. Centrifugation (3,000rev/minute for 10 minutes) yields multilammelar liposomes containingiron(III) maltol.

(1) In a modification of this procedure the phospholipid may be variedamong egg yolk phosphatidyl chlorine, dimyristoyl phosphatidylcholineand dipalmitoyl phosphatidylcholine together with a preparation ofcholesterol varying from 0 to 1 moles of cholesterol per mole ofphospholipid.

(B) A chloroform solution (2 ml) of the 3:1 neutral iron(III) maltolcomplex (5 mg/ml) is added to egg yolk phosphatidylcholine (100 mg) anda cholesterol (50 mg). The solution is rotary evaporated to yield a deepred skin on the surface of a round bottomed flask. Addition of 6 ml of abuffered solution of sodium chloride (100 mM, Tris.HCl: 20 mM, pH 7.4)followed by shaking for 15 minutes leads to a finely dispersedlipid-iron(III) maltol preparation. Centrifugation at 3000 revs/minutefor 100 minutes yields a liposome preparation which can be readilyfreeze dried. The entrapment of iron(III) maltol using this method isparticularly efficient.

Liposomes produced by either method are resistant to dissociation in thestomach but will undergo dissociation in the intestine.

Example 9 Tests with Iron Maltol in Human Subjects

Capsules containing 3:1 iron(III) maltol, together with an excess offree maltol were prepared according to the procedure of Example 7⁽¹⁾with the modification that at 3:1 molar ratio of maltol:iron was used(no free maltol therefore being present in the capsules) and that aproportion of the ferric chloride used in the preparation of the ironmaltol was radiolabelled as ⁵⁹Fe. The capsules each contained 10 mg ofiron, this amount being labelled with 1 microcurie of ⁵⁹Fe, and weresubjected to a single coating procedure.

(1) When preparing 3:1 iron(III) maltol on a large scale for inclusionin bulk preparation of capsules or other forms of compositions, it maybe advantageous to modify the procedure described in Example 7, and inExample 1, by carrying out the reaction in aqueous ethanol (4:1 v/vC₂H₅OH:H₂O) as the solvent and by adjusting the pH to 8.5 by theaddition of 2M aqueous sodium hydroxide rather than with solid sodiumcarbonate. Evaporation of the solvent after filtration will give aproduct containing some sodium chloride but this can be removed throughrecrystallisation of the 3:1 iron(III) maltol from water.

The test involved two normal subjects, i.e. not suffering from irondeficiency, one of whom took a 10 mg dose of iron (one capsule) and theother whom took a 20 mg does of iron (two capsules). The capsule orcapsules were taken after fasting overnight and fasting was thencontinued for a further 2 hours. The take-up of iron by the subjects wasfollowed by means of whole body counting, using a calibrated bodycounter, reading being taken 4 to 8 hours after ingestion of thecapsules to establish a base level and then after 7 and 14 days.Excretion of iron by the subjects was followed through the counting, ina large volume counter calibrated for iron, of daily urine samples over3 days and daily stool samples over 10 days.

The following results were obtained. The levels of take-up of iron asindicated by the whole body counting were substantially the same at 7days and at 14 days (apart from a small direction in the latter figuredue to decay of the ⁵⁹Fe rather than excretion thereof). For one subject(10 mg dose) the 14 day figure was 19% whilst for the other (20 mg dose)it was 11%. These figures were confirmed by the counting of stoolsamples (an essentially zero loss of iron being observed in the urine).

In an exactly similar experiment employing 10 mg and 20 mg of ⁵⁹Felabelled ferrous sulphate with the same subjects (at the same dosagelevel of iron) the level of take-up of iron, measured by whole bodycounting at 7 days, was 13% for one subject (10 mg dose) and 6% for theother (20 mg dose).

This test therefore showed a significantly increased uptake or iron inthese normal subjects from 3:1 iron(III) maltol as compared with ferroussulphate. An even greater difference would be anticipated for irondeficiency subjects.

We clam:
 1. A pharmaceutical composition comprising a compound being atissue permeable, neutral 3:1 hydroxypyrone:iron(III) complex of3-hydroxy-4-pyrone or of a 3-hydroxy-4-pyrone in which at least one ofthe hydrogen atoms attached to ring carbon atoms is replaced by analiphatic hydrocarbon group of 1 to 6 carbon atoms, wherein said complexis encapsulated by a material resistant to dissociation under aqueousacidic conditions.
 2. A pharmaceutical composition according to claim 1,in which the iron complex is encapsulated by a solid material which isresistant to dissociation under acidic aqueous conditions but which isadapted for dissociation under non-acidic aqueous conditions.
 3. Apharmaceutical composition according to claim 2 in capsule form.
 4. Apharmaceutical composition according to claim 2, in which each aliphatichydrocarbon group is an acyclic group of 1 to 4 carbon atoms.
 5. Apharmaceutical composition according to claim 2, in which each aliphatichydrocarbon group is an alkyl group.
 6. A pharmaceutical compositionaccording to claim 2, in which the compound is an iron complex of a3l-hydroxy-4-pyrone in which at least one of the hydrogen atoms attachedto ring carbon atoms is replaced by a substituent selected from thegroup consisting of methyl, ethyl, n-propyl and isopropyl.
 7. Apharmaceutical composition according to claim 6, in which eachsubstituent is a methyl group.
 8. A pharmaceutical composition accordingto claim 6, in which the substituted 3-hydroxy-4-pyrone has a singlesubstituent at the 2- or 6-position.
 9. A pharmaceutical compositionaccording to claim 2, in which the compound is the 3:1 iron complex of3-hydroxy-4-pyrone, 3-hydroxy-2-methyl-4-pyrone,3-hydroxy-6-methyl-4-pyrone or 2-ethyl-3-hydroxy-4-pyrone.
 10. Apharmaceutical composition according to claim 2, in which the compoundis the 3:1 iron complex of 3-hydroxy-2-methyl-4-pyrone.
 11. Apharmaceutical composition according to claim 2, in which the compoundis the 3:1 iron complex of 2-ethyl-3-hydroxy-4-pyrone.
 12. Apharmaceutical composition according to claim 2, in which the neutral3:1 complex is substantially free from complexes containing otherproportions of the hydroxypyrone and iron(III).
 13. A pharmaceuticalcomposition according to claim 2, in which the iron complex is insubstantially pure form.
 14. A pharmaceutical composition according toclaim 2 which additionally contains an iron chelating agent.
 15. Apharmaceutical composition according to claim 14, in which the ironchelating agent is uncomplexed 3-hydroxy-4-pyrone or an uncomplexed3-hydroxy-4-pyrone in which at least one of the hydrogen atoms attachedto ring carbon atoms is replaced by an aliphatic hydrocarbon group of 1to 6 carbon atoms, or a salt thereof containing a physiologicallyacceptable cation.
 16. A pharmaceutical composition according to claim15, which contains a complexed hydroxypyrone together with the samehydroxypyrone or a salt thereof in uncomplexed form.
 17. Apharmaceutical composition according to claim 16, which comprises the3:1 iron complex of 3-hydroxy-2-methyl-4-pyrone and uncomplexed3-hydroxy-2-methyl-4-pyrone or a salt thereof containing aphysiologically acceptable cation.
 18. A pharmaceutical compositionaccording to claim 2 which additionally contains folic acid.
 19. Amethod for the treatment of a patient to effect an increase in the levelof iron in the patient's bloodstream, which comprises administering tosaid patient in need thereof a composition according to claim 1, in anamount which is effective to achieve said increase.
 20. A method for thetreatment of a patient to effect an increase in the level of iron in thepatient's bloodstream, which comprises administering to said patient inneed thereof a composition according to claim 2, in an amount which iseffective to achieve said increase.
 21. A method according to claim 20,in which the compound is the 3:1 iron complex of a 3-hydroxy-4-pyrone,3-hydroxy-2-methyl-4-pyrone, 3 hydroxy-6-methyl-4-pyrone or2-ethyl-3-hydroxy-pyrone.
 22. A method according to claim 20 in whichthe compound is the 3:1 iron complex of 3-hydroxy-2-methyl-4-pyrone. 23.A method for the treatment of a patient to effect an increase in thelevel of iron in the patient's bloodstream which comprises administeringto said patient in need thereof a compound being a 2:1hydroxypyrone:iron (III) complex of 3-hydroxy-4-pyrone or of a3-hydroxy-4-pyrone in which at least one of the hydrogen atoms attachedto the ring carbon atoms is replaced by an aliphatic hydrocarbon groupof 1 to 6 carbon atoms, in an amount which is effective to achieve saidincrease.
 24. A method according to claim 23, in which each aliphatichydrocarbon group is an acyclic group of 1 to 4 carbon atoms.
 25. Amethod according to claim 23, in which each aliphatic hydrocarbon groupis an alkyl group.
 26. A method according to claim 23, in which thecompound is an iron complex of a 3-hydroxy-4-pyrone in which at leastone of the hydrogen atoms attached to ring carbon atoms is replaced by asubstituent selected from the group consisting of methyl, ethyl,n-propyl and isopropyl.
 27. A method according to claim 23, in whicheach substituent is a methyl group.
 28. A method according to claim 23,in which the substituted 3-hydroxy-4-pyrone has a single substituent atthe 2- or 6-position.
 29. A method according to claim 23, in which thecompound is a 2:1 iron complex of 3-hydroxy-4-pyrone,3-hydroxy-3-methyl-4-pyrone, 3-hydroxy-6-methyl-4-pyrone or2-ethyl-3-hydroxy-4-pyrone.
 30. A method according to claim 23, in whichthe compound is a 2:1 iron complex of 3-hydroxy-2-methyl-4-pyrone.
 31. Amethod according to claim 23, in which the compound is a 2:1 ironcomplex of 2-ethyl-3-hydroxy-4-pyrone.
 32. A method for the treatment ofa patient to effect an increase in the level of iron in the patient'sbloodstream, which comprises administering to said patient in needthereof a compound being a 1:1 hydroxypyrone:iron (III) complex of3-hydroxy-4-pyrone or of a 3-hydroxy-4-pyrone in which at least one ofthe hydrogen atoms attached to ring carbon atoms is replaced by analiphatic hydrocarbon group of 1 to 6 carbon atoms, in an amount whichis effective to achieve said increase.
 33. A method according to claim32 in which each aliphatic hydrocarbon group is an acyclic group of 1 to4 carbon atoms.
 34. A method according to claim 32, in which eachaliphatic hydrocarbon group is an alkyl group.
 35. A method according toclaim 32, in which the compound is an iron complex of a3-hydroxy-4-pyrone in which at least one of the hydrogen atoms attachedto ring carbon atoms is replaced by a substituent selected from thegroup consisting of methyl, ethyl, n-propyl and isopropyl.
 36. A methodaccording to claim 32, in which each substituent is a methyl group. 37.A method according to claim 32, in which the substituted3-hydroxy-4-pyrone has a single substituent at the 2- or 6-position. 38.A method according to claim 32, in which the compound is a 1:1 ironcomplex of 3-hydroxy-4-pyrone, 3-hydroxy-2-methyl-4-pyrone,3-hydroxy-6-methyl-4 -pyrone or 2-ethyl-3-hydroxy-4-pyrone.
 39. A methodaccording to claim 32, in which the compound is a 1:1 iron complex of3-hydroxy-2-methyl-4-pyrone.
 40. A method according to claim 32, inwhich the compound is 1:1 iron complex of 2-ethyl-3-hydroxy-4-pyrone.